Green synthesis of gold nanoparticles using Pelargonium Graveolens leaf extract: characterization and anti-microbial properties (An in-vitro study)

Background In recent years, there has been a notable increase in the level of attention devoted to exploring capabilities of nanoparticles, specifically gold nanoparticles AuNPs, within context of modern times. AuNPs possess distinct biophysical properties, as a novel avenue as an antibacterial agent targeting Streptococcus Mutans and Candida Albicans. The aim of this study to create a nano-platform that has the potential to be environmentally sustainable, in addition to exhibiting exceptional antimicrobial properties against Streptococcus Mutans as well as Candida Albicans. Methods this study involved utilization of Pelargonium Graveolens leaves extract as a cost effective and environmentally sustainable approach for the green synthesis of AuNPs. Subsequently, physicochemical characteristics were assessed employing a variety of analytical methods, including as transmission electron microscopy, X-ray diffraction, Field Emission Scanning Electron Microscope, Zeta potential, Ultraviolet visible absorption spectroscopy, and Energy dispersive X-ray spectroscopy and Fourier transform infrared spectroscopy. The antimicrobial efficacy against Streptococcus Mutans and Candida Albicans was evaluated. Nanoparticles of various shapes, including hexagonal, spherical, semi-spherical, and triangular, were synthesized. These nanoparticles exhibited a mean particle size of 294nm and demonstrated low degree of aggregation. These nanoparticles exhibited long-term stability and were capable of facilely combining with diverse bioactive compounds. Results The study demonstrated that AuNPs which is synthesized by green methods display potent antimicrobial properties. Conclusion Utilization of Pelargonium Graveolens AuNPs may exhibit a promising potential as an antibacterial agent against Streptococcus Mutans and Candida Albicans. Nanoparticles (NPs) have the potential to serve as a novel approach for addressing pathogen infections as well as for biomedical, dental and pharmaceutical purposes in the future.


Introduction
Nanotechnology has recently provided novel options for the treatment of several ailments.2][3][4] The comprehensive investigation of gold nanoparticles has surpassed that of other metal nanoparticles, owing to their comparatively minimal toxicity towards animal and microorganism cells in comparison to other metal nanoparticles. 5Gold nanoparticles display unique physicochemical characteristics in comparison to bulk solids, owing to their significantly larger surface area relative to their volume. 6The coloration of Gold nanoparticles is dependent on their structural characteristics, size, and level of aggregation.The application of red colloidal gold has been employed as a means of regeneration therapy.In addition, gold nanoparticles have been utilized in diagnostic and therapeutic drug delivery applications.The utilization of microorganisms, enzymes, and plant extracts has been proposed as a viable and environmentally conscious substitute for conventional chemical and physical techniques in the production of metallic nanoparticles.The utilization of the green synthesis approach has attracted significant interest as a cost-effective and eco-friendly methodology that facilitates the production of nanoparticles of diverse shapes and sizes, Demonstrating remarkable stability and compatibility. 7The study done by Lee et al.  investigates the efficacy of distinct active compounds derived from plants, particularly is flavones, protocatechuic acid, and Gallic acid, as reducing agents for the green synthesis of modified gold nanoparticles (AuNPs).The resulting nanoparticles exhibited remarkable stability and biocompatibility, with a lifespan of up to three months. 8In general, it has been observed that HAuCl 4 forms chlorine-carbon bonds with plant extracts. 9Furthermore, the plant extracts contain starch and glucose constituents that can serve as stabilizing and reducing agents during the synthesis of gold nanoparticles.This enables the nanoparticles to be stored for an extended period of 17 months while maintaining their stability. 10hankar et al in 2003 were able to synthesize gold nanoparticles (GNPs) of varying shapes through the utilization of Pelargonium graveolens leaves and its entophytic fungus.The plant Pelargonium graveolens and its entophyte have been recognized as potential candidates for the synthesis of gold nanoparticles.In particular, the researcher had conducted research on the reduction of aqueous chloroaurate ions using the leaves of the Pelargonium graveolens plant and an entophytic fungus. 11Due to its, antioxidant, antibacterial, antifungal, hypoglycemic, anti-inflammatory, and cancerfighting were only some of the reported biological and pharmacological effects of Pelargonium graveolens.Extracts from this plant are useful because they contain bioactive compounds.Citronellal, geranial, and linalool are three major bioactive components found in Pelargonium graveolens, which may explain their properties.In light of this, it is now recommended in herbal medicine to investigate the chemical components of this plant.In order to create safe and effective medications, it is crucial to understand the scientific foundation of their therapeutic activities. 12Nanoparticles hold distinct promise compared to their bulk equivalents.Since they are small and have a large surface area, they have demonstrated significant biological activity in the human body. 13The use of nanotechnology in dentistry has significant potential for transformative advancements.Nevertheless, it is crucial to acknowledge the potential risks associated with its misuse.The prioritization of applications within this discipline will ultimately be determined by factors such as time and human requirements. 14In recent years, there has been a significant increase in the utilization of nanoparticles in the formulation and advancement of various dental materials.This is mostly due to their ability to provide a distinctive

REVISED Amendments from Version 1
The main difference between this version and the previous version are the minor changes just italicized the following words (Pelargonium Graveolens, Streptococcus Mutans and Candida Albicans).
And change the affiliation to (Department of Pediatric and Preventive Dentistry, College of Dentistry, University of Baghdad, Iraq).
Any further responses from the reviewers can be found at the end of the article amalgamation of characteristics.The nanoparticles have a relatively large surface area to volume ratio in comparison to particles of equivalent substance due to their small size. 15The present study outlines a straightforward procedure for synthesizing AuNPs by employing Pelargonium Graveolens extract as an inexpensive and environmentally benign, stabilizing and reducing agent, furthermore, an evaluation was conducted on the physical characteristics and antimicrobial efficacy towards Streptococcus mutans and Candida albicans.

Microorganism
The study evaluated the antimicrobial effectiveness of the prepared Pelargonium Graveolens AuNPs against clinical isolates of Streptococcus mutans and Candida albicans.These isolates were obtained from the oral cavity of patients in the Pediatric and Preventive Dentistry Department at the University of Baghdad's College of Dentistry.The laboratory utilized standard biochemical methods to process and identifies these isolates.Following the transfer of the stock cultures onto Mueller-Hinton agar medium, an overnight incubation at 37°C was conducted, followed by storage at 4°C. Preparation of Pelargonium Graveolens leaf extract and Green synthesis of Pelargonium Graveolens AuNPs Pelargonium Graveolens leaves collection in Baghdad Governorate, Iraq.The collected leaves were thoroughly cleansed under running water to eliminate traces of chemicals and grime.After a thorough rinsing, the leaves of Pelargonium Graveolens were incubated overnight at 37 o C to dry.After incubation, the completely desiccated leaves were ground into a fine powder for use in the extraction process.A quantity of 100 g of air-dried ground plant material was subjected to extraction using aqueous alcohol as the solvent (methanol, water, ethanol (1:1:3) 80 % v/v) 500 mL the samples were subjected to Soxhlet extraction for a duration of 8 hours on a water bath.The extracts were subjected to solvent removal under reduced pressure at a temperature of 45°C using a rotary evaporator.Subsequently, the desiccated crude concentrated extracts were measured in order to determine the yield.Following this, the powder was diluted to achieve a concentration of 250 μg/ml, which corresponds to MIC (minimum inhibitory concentration) of the plant extract, after that, the extract was filtered 50ml of it was taken prepared for the purpose of using it with gold chlorides to act as a reducing agent in the preparation of gold nanoparticles.The gold ion solution was prepared in accordance with the methodology outlined by Ref. 16. with some modification, one gram of HAuCl 4 dissolved by 100 mL of DW to form a 10 mg/ml solution.Gold nanoparticles (Au NPs) were synthesized through a mix of a 1 mg/ml HAuCl 4 gold ion solution and a 50 ml Pelargonium Graveolens extract.The solution was subjected to a magnetic stirrer for duration of 30 minutes while being slightly heated to a temperature range of 35°C to 45°C.As a result, the mixture exhibited a quick alteration in color, transforming into a dark shade of purple/red within a brief time span.The observed change in color can be assigned to the formation of gold nanoparticles (AuNPs) with a concentration of 15.7 (ppm).The manifestation of a purple color following the mixture of the plant extract and HAuCl 4 solution signifies the production of Pelargonium Graveolens AuNPs.

Characterization of Pelargonium Graveolens AuNPs
An X-ray diffract meter (XRD-6000, SHEMADZU-Japan ®,™ ) used to evaluate the produced nanoparticles characteristics.(Current = 30 mA; voltage = 40 kV).A Cu-Kα incident beam (λ = 1.542A°) at 2θ = 20°-80°used to determine the patterns in which the particles diffracted.Transmission electron microscopy (PHILIPS CM-120-USA ®,™ ) is operating at 100 kV to evaluate the size and morphologies of the nanoparticles.Pelargonium Graveolens AuNPs solutions were characterized using a UV-Vis spectrophotometer (SHEMADZU1200i-Japan ®,™ ) the experiment employed a double beam UV-Vis spectrophotometer to observe the beams under different conditions within the spectral range of 200-1100 nm.An FTIR analysis was conducted utilizing the PerkinElmer Spectrum-USA instrument, covering spectral ranges from 4500 to 500 cm -1 .FTIR analysis was conducted in order to examine the functional groups of biomolecules that are responsible for capping, viable stabilization, and reduction of the prepared Pelargonium Graveolens AuNPs.A Zeta potential test was conducted using a Zetasizer Nano ZS (Malvern Instruments ®,™ ) at a temperature of 25°C.The (Zetasizer 6.01 software ®,™ )was employed for data analysis.The purpose of the zeta-potential measurements was to examine the stability of Pelargonium Graveolens AuNPs in the suspension.Field Emission Scanning Electron Microscopy (FESEM) employed utilizing a (ZIESS, Germany ®,™ ) Electron Microscopy to identify the morphological features of Pelargonium Graveolens AuNPs.The investigation of elemental composition was carried out using EDX with a (Thermo Scientific TM NORANTM ®,™ System 6 EDS system).

Biological activities of Pelargonium Graveolens AuNPs Antibacterial efficacy of Pelargonium Graveolens AuNPs
The antimicrobial effect of Pelargonium Graveolens AuNPs was investigated using the Agar diffusion method.Ten isolates of each S. mutans and C. Albican were applied separately on MH agar plates; the previous approach was explained by Ref. 17. Mueller-Hinton agar media was poured separately into sterile petri dishes and wait until setting.A volume of 0.1ml of activated Streptococcus mutans and Candida Albican was evenly distributed onto Mueller-Hinton agar plates and allowed to incubate at the room temperature for duration of 10 minutes.Subsequently, using a sterile stainless steel Cork borer, uniform and sufficiently deep wells measuring 6mm in diameter were generated in the Mueller-Hinton agar.This process was repeated six times on each plate and for each microorganism.Each well was filled with 0.2 mL of the agent to be tested at varying concentrations of 0.06, 0.12, 0.25, and 0.5 mg/mL.The last well filled with Chlorhexidine 0.12% as a positive control, while the center well filled deionized water as a negative control.Plates left at the room temperature for 10 minutes and then incubated aerobically for 24hrs at 37C.Each zone of inhibition was measured across the diameter of each well.No zone of inhibition indicated complete resistant of S. mutans and C. Albican a to the test agent.Then, we calculated the MIC, MBC, and MFC of Pelargonium Graveolens AuNPs against the individual microorganisms by agar dilution method against S. mutans, and C. albicans.Pelargonium Graveolens AuNPs concentrations of 0.06, 0.12, 0.25, 0.5 mg/ml were serially diluted in Mueller-Hinton agar to make 10 mL of agar and extracts, which were then placed into Petri dishes and allowed to solidify before being inoculated with 0.1 ml of activated isolates of microorganisms.All of these Petri dishes, including the control plates, were incubated at 37°C overnight (the negative control, which contained Mueller-Hinton agar with microbial inoculums without the addition of the extracts, and the positive control plates, which contained MH-A and different concentrations of the Pelargonium Graveolens AuNPs without microbial inoculums).The presence or lack of bacteria growth on the plates is checked.The MIC of Pelargonium Graveolens AuNPs is the concentrations at which microbial growth is totally inhibited.The identical approach was followed for all of the microbial isolates, that had been described previously. 18The MBC/MFC of Pelargonium Graveolens AuNPs is the concentrations at which microbial growth is totally killed.

Statistical analysis
Statistical Package for Social Research was used for descriptive analysis, and presentation (SPSS Statistical Package for social Science version-22, Chicago, Illinois, USA).The Levene test and Shapiro-Wilk test were conducted on a quantitative variable, which encompassed the minimum, maximum, mean, standard deviation (SD), and standard error (SE).The One-way Analysis of Variance (ANOVA) statistical method was employed, along with Tukey's Honestly Significant Difference (Tukey's HSD) and Dunnett's T3 posthoc tests, to conduct multiple pairwise comparisons among the groups.The results are considered statistically insignificant when the P value is greater than 0.05, while they are deemed statistically significant when the P value is less than 0.05.

Characterization of prepared nanoparticle Determination of the shape and size
Transmission electron microscopy (TEM) was employed to investigate the characterization of the synthesized nanoparticles (NPs).The morphology of gold nanoparticles (AuNPs) is characterized by a combination of hexagonal, spherical, semi-spherical, and triangular-shaped particles.The size and abundance of gold nanoparticles particles decrease as the concentration of Pelargonium Graveolens leaves extracts increases.The morphology Pelargonium Graveolens AuNPs is depicted in Figure 1-A, where the AuNPs are represented as a core and the Pelargonium Graveolens acts as a shell surrounding the AuNPs surface, and as shown in Figure 1-B the average size of Pelargonium Graveolens AuNPs has been estimated to be 294 nm due to the small concentration of Pelargonium Graveolens leaves extracts is 250 μg/ml (MIC of plant extract), It is considered a good and safe particle size because the smaller size easily to uptake by mammalian cells this is agree by Ref. 19.

XRD analysis
For further determination of the Pelargonium Graveolens AuNPs, The X-ray diffraction (XRD) pattern was utilized to analyze the diffraction peaks observed at 2θ = 39.9°,44°, and 65.28°, which can be attributed to the 111, 200, and 220 crystallographic planes of the cubic structure of the gold nanoparticles (Au NPs) as depicted in Figure 2.These results are consistent with the data provided by the Joint Committee on Powder Diffraction Standards (JCPDS) under the reference number 001-1172.The results mentioned above agree with the data presented by Ref. 20.The current data indicates that XRD pattern of the Pelargonium Graveolens AuNPs closely resembles that of pure gold nanoparticles.The mean size of nano crystallites in Pelargonium Graveolens AuNPs is 69.587 nm calculated From the well-known Scherrer formula (D = 0.9λ/β.Cos θ) In this context, the symbol D represents the average size of crystallites, β refers to the line broadening as quantified by the full width at half maximum (FWHM) of a peak, λ represents the wavelength of X-rays used for irradiation, and θ signifies the maximum position value of the peak 21 as shown in Table 1.

FTIR spectra of prepared NPs
In the cases of Pelargonium Graveolens AuNPs the FTIR measurements detailed in Table 2 were conducted in an effort to determine the character of the organic protection layer surrounding the AuNPs based on comparable research in the literature. 22,23The observed significant shifts in the spectra of certain peaks subsequent to the formation of AuNPs are   hypothesized to be attributed to the impact exerted by the adjacent metal surface.Hence, it is postulated that the observed displaced peaks, as documented in Table 2, are indicative of the presence of organic matter surrounding the synthesized AuNPs.Figure 3  Furthermore, the IR band at 1636.44 cm -1 is characteristic of the C=O expanding of the carboxylic group.Hence, it is possible that enzymes/proteins are involved in the process of reducing metal ions through the oxidation of aldehydes to carboxylic acids.Proteins possess the capacity to form links with gold nanoparticles (AuNPs) via carboxylate ions found in amino acid residues or through free amine groups within the protein's structure. 24,25In addition, the presence of the C=O stretching mode indicates the presence of the carboxylic acid (-COOH) group in the material that is attached to gold nanoparticles (Au NPs).Hence, the spectral peak observed at a wavenumber of 1636.44 cm -1 can be attributed to the manifestation of amine groups, signifying a possibility of protein binding onto gold nanoparticles.The provided information holds significant value in informing the design of functionalization procedures, particularly in the context of utilizing particles for drug delivery purposes.

UV-Vis absorbance spectrum
Figure 4 shows the absorbance of the peak of Pelargonium Graveolens AuNPs was at 527 nm.Pelargonium Graveolens AuNPs show a massive peak.The successful synthesis of Pelargonium Graveolens AuNPs was confirmed by the observation of a dark purple color in the reaction mixture and the presence of specific absorption spectra at a wavelength of 527 nm in the UV-vis absorption spectroscopy.This outcome aligns with findings reported in previous studies. 16

Zeta potential
The zeta potentials of the particles present in the sample of Pelargonium Graveolens AuNPs were determined at a temperature of 25°C.Figure 5 shows that the Pelargonium Graveolens AuNPs sample showed positive zeta-potential values between 0 and +50 mV the observed range can be classified as an initial state of stability for colloidal systems that agree with. 26The zeta potential of the Pelargonium Graveolens AuNPs was measured to be 27 mV Figure 5.This value suggests that the nanoparticles were stable and had a reduced propensity to aggregate and form larger particles.

FESEM-EDX analysis of particle composition
The results clearly presented in Figure 6-B demonstrate that the particle composition unequivocally corresponds to gold.The presence of trace amounts of additional elements, such as oxygen (O), was observed in the elemental composition of the plant extract after the solvent evaporation process.The elemental composition of the plant extracts was determined using inductively coupled plasma optical emission spectroscopy (ICP-OES), revealing the presence of trace amounts of these elements, among others.The data exhibited a strong correlation between the concentrations of different elements present in the plant extracts and the results obtained from Energy-Dispersive X-ray Spectroscopy (EDX), as seen previously in this report, 16 and the findings from field emission scanning electron microscopy (FESEM) analysis, as depicted in Figure 6-A, indicate that the gold nanoparticles (AuNPs) were effectively separated and did not form aggregates.This can be attributed to the presence of Pelargonium Graveolens, which functions as a protective agent, corroborating the observations made through transmission electron microscopy (TEM) and this agree with. 27

Biological studies Antibacterial activity of Pelargonium Graveolens AuNPs
The antimicrobial efficacy of Pelargonium Graveolens AuNPs was evaluated using Streptococcus mutans and Candida albicans as test organisms.Significant zones of inhibition were observed subsequent to the exposure of the microorganisms to the Pelargonium Graveolens AuNPs that were prepared Figure 7A,B.The findings of the present study demonstrate the effectiveness of the synthesized nanoparticles in inhibiting the growth rate of Streptococcus mutans and Candida albicans following a 12-hour exposure period as shown in Figure 8.

Determination of Minimum Bactericidal Concentration (MBC)
The study results indicate that MBC for Candida albicans was 0.5 mg/ml, while MBC for Streptococcus Mutans was 0.12 mg/ml, as presented in the Tables 3 and 4. Subsequent re-culturing on BHI-agar media revealed no growth at these concentrations as shown in Figure 8 indicating that the 0.5 mg/ml concentration had a fungicidal effect on Candida albicans, while the 0.12 mg/ml concentration had a bactericidal effect on Streptococcus Mutans.Also the results presented in the table indicate that the MIC for Candida albicans was 0.25 mg/ml, while for Streptococcus Mutans it was 0.06 mg/ml.However, onto re-culturing on BHI-agar, it was observed that these concentrations exhibited growth, indicating that they had a bacteriostatic effect.

Discussion
The results show that with increase concentration of Pelargonium Graveolens AuNPs, the diameter of inhibition zone of streptococcus mutans growth increased to reach the maximum diameter as shown in Table 5 and Figure 9.
And the results show that with increase concentration of Pelargonium Graveolens AuNPs, the diameter of inhibition zone of Candida Albicans growth increased to reach the maximum diameter as shown in Table 6 and Figure 10.
In this study although the mean inhibition zone for Streptococcus mutans growth by Pelargonium Graveolens AuNPs (1.16 cm) in concentration 0.06 mg/ml is lower than that of Chlorhexidine gluconate 0.12% (2.17 cm), but this mean    inhibition zone for Streptococcus mutans growth by Pelargonium Graveolens AuNPs significantly increases with increasing concentration till it reaches its highest value 2.18 cm at a 0.5 mg/ml concentration as shown in Table 5 and Figure 9.The study found that Pelargonium Graveolens AuNPs at a concentration of 0.5 mg/ml exhibited a mean inhibition zone of 1.79 cm for Candida Albican growth, which was lower than the mean inhibition zone of 1.95 cm, observed for Chlorhexidine gluconate 0.12%.However, it is possible that the mean inhibition zone for Candida Albican growth by Pelargonium Graveolens AuNPs may increase with higher concentrations as shown in Table 6 and Figure 10.This finding indicates that the antibacterial effect of Pelargonium Graveolens AuNPs is concentration dependent.Due to there is no previous studies on the effect of Pelargonium Graveolens AuNPs against Streptococcus mutans and Candida albicans, so that the study compared with other studies of the effects Pelargonium Graveolens AuNPs or effect of the secondary metabolites of Pelargonium Graveolens on others microorganisms.This finding may be attributed to the synergistic or additive effect of the secondary metabolites that have antimicrobial activity such as citronellol that have antimicrobial and anti-oxidant activity, 28 and the research conducted by Guimares et al., 2019 discovered that citronellol exhibited significant bactericidal properties against S. aureus strains. 29And it agree with Friedman et al., 2004 have classified essential oils and their compounds into two categories, namely slow-acting and fast-acting compounds.The authors have reported that terpineol, eugenol, geranial, carveol, and citronellol are categorized as fast-acting compounds due to their ability to rapidly inactivate microorganisms such as E. coli and S. Typhimurium within a short duration of 2 hours.According to reports, certain antimicrobial agents such as carvacrol, cinnamaldehyde, and geraniol have been identified as fast-acting compounds capable of inactivating microorganisms such as E. coli and S. Typhimurium within a five-minute timeframe. 30Geraniol and citronellol are an active component of Pelargonium Graveolens. 28Additionally it may be due to the presence of certain compounds in Pelargonium Graveolens AuNPs which might exert their effect either on the cell membrane of streptococci mutans or on the enzymes that are necessary for the growth of this bacteria and this assumption need further studies for its confirmation.

Raya Abid
Ibnsina university of medical and pharmaceutical sciences, Baghdad, Iraq The article is scientific and original.It's necessary to find new approaches in the prevention of diseases and the research was trying to find these approaches.Some minor revisions are necessary before the duplication as follow: Abbreviations should be removed from abstract In abstract.
○ Always starting should be done by capital letters.

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Reference for the isolation and preparation of microorganisms should be added.

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In method, MIC and other abbreviations are not clear.Reviewer Expertise: preventive dentistry diverse shapes and dimensions of Au NPs.The successful synthesis of stable AuNPs has been supported by a range of characterization methods, including UV-Vis, Zeta potential, FESEM, EDX, XRD, TEM, and FTIR.The green synthetic gold nanoparticles form Pelargonium Graveolens leaves extract show low aggregation degree were obtained, exhibiting longterm stability.These nanoparticles can readily associate with diverse bioactive compounds.In addition, it is possible for Au NPs to combine with diverse bioactive compounds that adhere to the Pelargonium Graveolens extract, such as protein-based molecules, alkynes and phenolic compounds.This amalgamation could potentially enhance the biological activity of Au NPs that demonstrated effective antibacterial properties against Streptococcus Mutans and Candida Albicans in comparison with Chlorhexidine, while also maintaining a good level of safety.According to these results it can be considered as promising oral health care product in the future and it can be used as mouthwash as alternative of Chlorhexidine mouthwash that still had adverse effects over time.) Competing Interests: No competing interests were disclosed.
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Figure 1 .
Figure 1.A -TEM of Pelargonium Graveolens AuNPs B -Distribution of particle size of Pelargonium Graveolens AuNPs.
demonstrates the Fourier Transform Infrared (FTIR) analysis conducted to assess the significant biomolecules involved in the capping and stabilization of Pelargonium Graveolens AuNPs synthesized through a green method.Intense absorptions are observed at 3436.59, 2067.21,1636.44, and 683.21 cm -1 .IR band at 3436.59 cm -1 refers to the OH stretching mechanism of the proteins, polyphenols, and carbohydrates.The band noticed at 2067.21 and 683.21 cm -1 related to the CC expanding of the alkynes group and C-N/C-Cl in plane bending, respectively.

Figure 9 .
Figure 9. Mean diameter on inhibition zone of streptococcus mutans.

Figure 10 .
Figure 10.Mean diameter on inhibition zone of Candida Albican.

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It's necessary to check the grammar and not use the pronoun 'we'.

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Its necessary not to start with abbreviations like FTIR when you say previous studies, you have to put them and not just one.○Finallysummarize the conclusion should be more clear and precise.....best regard○Is the work clearly and accurately presented and does it cite the current literature?YesIs the study design appropriate and is the work technically sound?YesAre sufficient details of methods and analysis provided to allow replication by others?YesIf applicable, is the statistical analysis and its interpretation appropriate?YesAre all the source data underlying the results available to ensure full reproducibility?YesAre the conclusions drawn adequately supported by the results?YesCompeting Interests: No competing interests were disclosed.

Table 5 .
Descriptive and statistical test of DIZ of S. Mutans among groups.

Table 3 .
Determination of MIC and MBC against Streptococcus Mutans.

Table 4 .
Determination of MIC and MBC against Candida albicans.

Table 6 .
Descriptive and statistical of DIZ of Candida Albican among groups.ConclusionTo our knowledge, the current investigation represents a novel contribution to the scientific literature, as it is the first to demonstrate the antimicrobial efficacy of Pelargonium Graveolens synthesized through green synthesis techniques against Streptococcus Mutans and Candida Albicans.There exists a notable degree of interest in the investigation of potential biomedical applications associated with gold nanoparticles (Au NPs).The current investigation employed extracts of Pelargonium Graveolens for creating diverse shapes and dimensions of Au NPs.The successful synthesis of stable AuNPs has been supported by a range of characterization methods, including UV-Vis, Zeta potential, FESEM, EDX, XRD, TEM, and FTIR.The composition consists of a combination of hexagonal, spherical, semi-spherical, and triangular like particles nanoparticles with a particle size mean 294nm and low aggregation degree were obtained, exhibiting long-term stability.These nanoparticles can readily associate with diverse bioactive compounds.A positive zeta potential value indicates favorable stability of the particles in the suspension.In addition, it is possible for Au NPs to combine with diverse bioactive compounds that adhere to the Pelargonium Graveolens extract, such as protein-based molecules, alkynes and phenolic compounds.This amalgamation could potentially enhance the biological activity of Au NPs.The Pelargonium Graveolens AuNPs that were synthesized using green methods have demonstrated effective antibacterial properties against Streptococcus Mutans and Candida Albicans in comparison with Chlorhexidine, while also maintaining a good level of safety.According to these results it can be considered as promising oral health care product in the future and it can be used as mouthwash as alternative of Chlorhexidine mouthwash that still had adverse effects over time.Research article on Green synthesis gold nanoparticles from P. graveolens and their antimicrobial activity is exciting work.First, the author must change the microbial strains and plant name in italics form where it is mentioned.Second, Previous literature work is given in the form of a reference number but it should be in author-name.Statistical analysis P value should be written in the symbol form P>0.05 or P<0.05.

the work clearly and accurately presented and does it cite the current literature? Yes Is the study design appropriate and is the work technically sound? Yes Are sufficient details of methods and analysis provided to allow replication by others? Yes If applicable, is the statistical analysis and its interpretation appropriate? Partly Are all the source data underlying the results available to ensure full reproducibility? Yes Are the conclusions drawn adequately supported by the results? Yes Competing Interests:
No competing interests were disclosed.

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.
This is an open access peer review report distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.